Foot-and-mouth disease (FMD) is an economically devastating infectious disease of farm animals. Cattle, pigs, sheep and goats are all susceptible. The causative agent is FMDV, an aphthovirus of the Picornaviridae family for which seven serotypes have been described. The FMDV genome consists of a single RNA positive strand encoding four structural proteins (VP1-VP4) and, at least, ten non-structural polypeptides induced in infected cells. The non-structural proteins include a viral RNA replicase 3D (Newman et al., Proc Natl Acad Sci USA, 1994, 91:733), also termed FMDV infection associated antigen (VIAA). The coding region for structural and nonstructural proteins is shown schematically in FIG. 1.
Animal husbandry is a major industry which can be crippled by the spread of FMDV through the susceptible population. The presently available method for controlling the disease is by vaccination with a chemically inactivated viral composition. Despite the availability of the vaccine, it is very important when there is a outbreak of the disease to be able to identify rapidly the animals that have been infected to isolate them from the non-diseased animals. Moreover, it has been found that the infected but convalescent animals frequently become carriers of the virus. Such animals can become the source of new outbreaks of the disease and can introduce antigenic variants into a susceptible population. Because of this problem, a rapid serological method is needed to identify previously infected but convalescent and asymptomatic animals and distinguish them from vaccinated animals. Such a method would aid regional efforts to eliminate the virus through vaccination programs, and serve to facilitate international livestock trade even in the event of an outbreak of the disease.
The detection of VIAA (also known as 3D) by agar gel immunodiffusion or virus isolation have been used for years to identify infected animals. The low sensitivity of these methods makes them difficult for routine application. To overcome this problem, ELISA assays to detect specific antibodies against the whole virion (Hamblin et al., J Immunol Methods, 1986, 93:115), viral subunits (Smitsaart et al., J Virol, 1986, 57:983) or VIAA (Alonso et al., Prev Vet Med, 1990, 9:233) have been developed. The tests which detect the antibodies to the whole virus or to the structural proteins cannot distinguish between infected and vaccinated animals. The VIAA-based assays also failed to distinguish between infected and vaccinated animals because the infected cell culture extracts used in vaccines usually contain the 3D protein at a concentration sufficient to provoke antibodies against 3D (Pinto et al., J Hyg Camb, 1979, 82:41).
It has been reported that non-structural proteins 2B, 2C, 3AB, and 3ABC, produced in BHK cells infected with the virus, are precipitated by sera from convalescent animals, but only occasionally by those that have been vaccinated (Villiner et al., Veterinary Microbiol, 1989, 20:235; Tesar et al., Vir Genes, 1989, 3:29; Berger et al., Vaccine, 1990, 8:213; Neitzert et al., Virology, 1991, 184:799; Bergmann et al., Am J Vet Res, 1993, 54:825; Rodriguez et al., Arch Virol, 1994, 136:123; Lubroth et al., Res Vet Sci, 1995, 59:70). The use of 2C and 3ABC proteins have been proposed as potential candidates for the differential diagnosis of convalescent and vaccinated animals. However, 3ABC-derived polyproteins have proven to be unreliable for differential diagnosis (Lubroth et al., 1995). Moreover, the existing methods based on the detection of 2C and 3ABC proteins and polyproteins are expensive and cumbersome and impractical for routine assays. The assay methods, such as immunoprecipitation (Lubroth, et al., 1995); determinations of relative reactivities (i.e., ratios between different ELISA procedures); and immunoblots (Rodriguez et al., 1994) suffer from poor sensitivity and lack of specificity. It has also been proposed to use peptides derived from 2C and 3ABC in immunoassay. However, up to the present none have been reported.
Synthetic peptides have been used in the recent years to map antigenic or immunogenic sites on the surface of proteins, an approach also known as "site-directed-serology". This approach has been explored by one of the co-inventors (Wang, C. Y.) to identify and characterize highly antigenic epitopes on various viral proteins of HIV, HTLV and HCV and to develop sensitive and specific diagnostic immunoassay using peptides comprising these antigenic epitopes for HIV, HTLV and HCV (U.S. Pat. Nos. 4,735,896, 4,879,212, 4,833,071, 5,476,765, 5,106,726, 5,436,126). These assays have provided excellent sensitivity and specificity due to the high molar concentration of the reagent and the lack of unrelated bacterial, viral or host cell proteins. Further, in the case of HTLV-I and HTLV-II, the peptide-based immunoassay has provided an unmatched capability to differentiate between two closely related viruses. These peptide-based immunoassay overcame many of the existing problems associated with either viral lysates or recombinantly produced proteins.
The present invention employs novel FMDV nonstructural protein derived peptides and peptide compositions identified through site directed serology as immunoreagents. The method overcomes the deficiencies in sensitivity and specificity of the currently available FMDV-immunoassay. The immunoassays incorporate the peptides of the present invention for the capture of antibodies to Foot-and-mouth Disease Virus (FMDV) in animal body fluids and are useful for the diagnosis of FMDV infection, to identify potential carrier status, and to differentiate infected from vaccinated animals.